The overall objectives of this project during the last l9 years have been to study the nature of glycolytic enzymes, with particular emphasis on rate limiting enzymes, and how hormones and other chemical agents affect their activity. We are proposing to extend our studies on one of the most critical enzymes in th regulation of glycolysis in the mammalian heart, phosphofructokinase (PFK)>. Our philosophy is that a better knowledge of the regulation of this enzyme will contribute to our understanding of the molecular mechanisms of action of different hormones that affect glycolysis such as epinephrine and insulin. We propose (a) to continue our studies on the chemical nature of the allosteric sites in PFK through chemical modification by affinity label reagents and group specific reagents, (b) to examine the role of phosphorylation and dephosphorylation on the regulation of PFK, (c) to study the relationship between the degree of PFK aggregation and its catalytic activity, (d) to examine the nature of soluble and particulate PFK and how a mechanism involving reversible conversion of these two forms may take part in the regulation of glycolysis, (e) to study the molecular effects of epinephrine on muscle PFK, and (f) to study the regulation of PFK in "glycogen particles" isolated from the heart and to study the complementarity in the regulation of glycolytic enzymes manifested in these particles. Our studies on PFK will be performed on the enzyme purified and crystallized from the sheep heart. Physiological experiments will be carried out on extracts from the heart, the skeletal muscle, or "glycogen particles" isolated from the skeletal muscle. Since PFK is involved in the "Pasteur effect", our studies should contribute to our understanding of the biochemical regulatory mechanisms involved during cardiac anoxia and of how the cardiac muscle can control energy production to meet the needs for high energy.